Recombinant antibacterial group IIA phospholipase A2 and methods of use thereof

ABSTRACT

Disclose herein is a novel recombinant mutant protein of human Group IIA phospholipase A (PLA2) which has enhanced antibacterial activity when compared to the wild-type human Group IIA PLA2, pharmaceutical formulations comprising the protein and methods of use thereof. Additionally, the formulations may comprise other bioactive compounds, such as, e.g., conventional antibiotics, that act additively or synergistically with Group IIA PLA2 in order to promote bacterial killing.

This patent application claims the priority of U.S. provisional patentapplication No. 60/172,467, filed on Dec. 17, 1999, which isincorporated herein by reference.

This application claims priority under 35 U.S.C. §119 to provisionalapplication No. 60/172,467 filed Dec. 17, 1999.

The United States Government has certain rights to this invention byvirtue of funding received from the U.S. Public Health Service GrantRO1-A1 18571.

This application claims priority under 35 U.S.C. §119 to provisionalapplication No. 60/172,467 filed Dec. 17, 1999.

FIELD OF THE INVENTION

This invention pertains to a novel recombinant mutant protein of humanGroup IIA Phospholipase A2 (PLA2) which has significantly enhancedantibacterial activity compared to the wild-type human Group IIA PLA2,pharmaceutical formulations comprising the protein and methods of usethereof.

BACKGROUND OF THE INVENTION

The growing prevalence of antibiotic resistance in bacterial pathogenshas stimulated renewed interest in the discovery of novel antibiotics.U.S. Pat. No. 5,874,079 discloses that a “Group IIA” 14 kDaPhospholipase A2 (PLA2), mobilized during inflammation expresses potentbactericidal activity toward a broad range of clinically importantGram-positive bacteria and enhances the activity of the host defensemechanisms toward many Gram-negative bacteria.

The phospholipase A2 (PLA2) family of enzymes hydrolyze the sn-2 esterof glycerophospholipids to produce a fatty acid and a lysophospholipid(Dennis, J.Biol. Chem. 269:13057-13060, 1994; Gelb et al, Ann. Rev.Biochem. 64, 653-688, 1995; Waite, The phospholipases, Plenum Press, NewYork, 1987). Based on amino acid sequences, 10 groups of PLA2s have beenidentified, including eight from mammals (Dennis, Trends Biochem. Sci.22: 1-2, 1997; Cupillard et al., J. Biol. Chem. 272: 15745-15752, 1997).Group IIA PLA2 in mammals are produced by many different cell typesincluding phagocytic cells, platelets, Paneth cells and lacrimal cells.It has been shown that both rabbit and human Group IIA PLA2 can, inconcert with other host defense mechanisms, increase the destruction ofgram-negative bacteria (Wright et al., J. Clin. Invest. 85: 1925-1935,1990; Weiss et al., J. Biol. Chem. 269: 26331-26337, 1994 Elsbach etal., Trends Microbiol. 2: 324-328, 1994 and Madsen et al., Infect.Immun. 64: 2425-2430, 1996) and by itself, kill many gram-positivebacteria (Weinrauch et al., J. Clin. Invest. 97: 250-257, 1996). Theantibacterial activity of Group IIA PLA2 appears to be a specificattribute of the mammalian 14 kDa isoform. This is further exemplifiedin experimentally induced local inflammatory (ascitic) fluid in rabbits,whereby the mobilization of Group IIA PLA2 is fully responsible for thepotent bactericidal activity expressed in the fluid toward S. aureus andseveral other gram-positive bacteria (Weiss et al., en supra). Normalplasma, by contrast contains low levels of PLA2 and antistaphylococcalactivity. It has recently been shown that the mobilization of thisenzyme in baboons during inflammation may play an important role in hostdefense mechanisms against invading bacteria (Weinrauch et al., J. Clin.Invest. 102 (3): 633-638, 1998).

In biological fluids, as little as 100 ng/ml of the human Group II APLA2 is sufficient to kill greater than 99% of 10⁶ Staphylcoccus aureuscells/ml, including all multi-drug resistant clinical isolates tested.The bactericidal activity of the PLA2 was dependent on catalyticactivity and was enhanced synergistically by the co-treatment withsub-inhibitory doses of β-lactam antibiotics. The potent antibacterialactivity of the mammalian Group IIA PLA2 is not expressed by otherclosely related PLA2s reflecting the presence and localization of a highdensity of basic residues in the Group IIA PLA2 that is absent in allother subsets of related PLA2s.

U.S. Pat. No. 5,874,079 discloses that the rabbit Group IIA PLA2possesses 10 fold greater antibacterial activity than the human enzyme.Since it is preferable to treat humans with human-derived therapeuticproteins, what is needed is a human PLA2 with activity similar to therabbit counterpart.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for treatingGram-positive bacterial infections in humans, by administeringbactericidal-effective amounts of mutant human Group IIA PLA2.

In another aspect, the invention provides pharmaceutical formulationshaving bactericidal activity against Gram-positive bacteria. Theseformulations comprise bactericidal-effective concentrations of mutanthuman Group IIA PLA2 and a pharmaceutically acceptable carrier ordiluent. Additionally, the formulations may comprise other bioactivecompounds, such as, e.g., conventional antibiotics, that act additivelyor synergistically with Group IIA PLA2 to promote bacterial killing.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in light of the present description,claims and drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1(A, B, C and a,b,c) are space-filling models of wild-type human(FIGS. 1A, a), rabbit (FIGS. 1B, b) and mutant (G72K.T103K, FIGS. 1C, c)human Group IIA PLA2 showing the distribution of charged residues withinthese PLA2. Models are based on the solved X-ray structure of humanGroup IIA PLA2 (Scott, D. L., White, S. P., Browning, J. L., Rosa, J.J., Gelb, M. H., Sigler, P. B. 1991. Science 254: 1007-1010). Sitesshown in black represent basic residues (arg and lys), sites in graycorrespond to acidic residues (asp and glu). All other residues aredisplayed in white. Two images of each enzyme are shown corresponding toapproximately 180 degree rotations (a, b, c), for WT human, WT rabbitand mutant human, respectively. Note that residues are numbered in acontinuous fashion according to the primary structures of these PLA2s.

FIG. 2 is a graphic illustration of the comparison of bactericidalactivity of native human and rabbit Group II A PLA2 towards S. aureus52A.

FIGS. 3(A and B) show the DNA (A), SEQ. ID. NO. 1 and the protein (B),SEQ. ID. NO. 2 sequence, respectively, of mutant human Group IIA PLA2.Residues differing from sequence of wild-type human Group IIA PLA2 areshown in bold and underlined.

FIG. 4 is a graphic illustration of the comparison of the bactericidalactivity of human wild-type (WT) and mutant human and WT rabbit Group IIA PLA2 toward S. aureus RN450.

FIG. 5 is a graphic illustration of the comparison of the bactericidalactivity of human wild-type (WT), and mutant human and WT rabbit GroupII A PLA2 towards S. aureus RN450 and two clinical isolates, strains 5Aand 18 S. aureus.

FIGS. 6(A, B and C) are graphic illustrations showing WT human and WTrabbit and mutant human Group II A PLA2-induced killing of S. aureusstrains expressing 1(A) 5(B) or 8(C) capsular serotype polysaccharides.

FIGS. 7(A and B) is a graphic illustration showing that encapsulated (A)and non-encapsulated (B) S. aureus were equally susceptible to killingby a whole inflammatory fluid and that killing of both strains wasprevented by neutralizing antiserum to Group IIA PLA 2.

FIG. 8 is a graphic illustration showing that the killing ofencapsulated S. aureus by whole inflammatory exudates is blocked byGroup IIA PLA2 antiserum.

FIG. 9 is a graphical illustration showing that the mutant human GroupIIA PLA 2 enzyme accelerates clearance of S. aureus infection in vivo.

FIG. 10 is a graphical illustration showing that the mutant human GroupII A PLA 2 reduces S. aureus bacteremia.

FIG. 11 is a graphical illustration showing that administration of themutant human Group IIA PLA 2 reduces metastatic kidney infection causedby S. aureus.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents, and literature references cited inthis specification are hereby incorporated by reference in theirentirety.

Definitions

1. “Nucleic acid” or “polynucleotide” as used herein refers to purine-and pyrimidine-containing polymers of any length, eitherpolyribonucleotides or polydeoxyribonucleotides or mixedpolyribo-polydeoxyribo nucleotides. This includes single- anddouble-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids,as well as “protein nucleic acids” (PNA) formed by conjugating bases toan amino acid backbone. This also includes nucleic acids containingmodified bases.

2. An “isolated” nucleic acid or polypeptide as used herein refers to anucleic acid or polypeptide that is removed from its originalenvironment (for example, its natural environment if it is naturallyoccurring). An isolated nucleic acid or polypeptide contains less thanabout 50%, preferably less than about 75%, and most preferably less thanabout 90%, of the cellular components with which it was originallyassociated.

3. A nucleic acid or polypeptide sequence that is “derived from” adesignated sequence refers to a sequence that corresponds to a region ofthe designated sequence. For nucleic acid sequences, this encompassessequences that are homologous or complementary to the sequence, as wellas “sequence-conservative variants” and “function-conservativevariants.” For polypeptide sequences, this encompasses“function-conservative variants.” Sequence-conservative variants arethose in which a change of one or more nucleotides in a given codonposition results in no alteration in the amino acid encoded at thatposition. Function-conservative variants are those in which a givenamino acid residue in a polypeptide has been changed without alteringthe overall conformation and function of the native polypeptide,including, but not limited to, replacement of an amino acid with onehaving similar physico-chemical properties (such as, for example,acidic, basic, hydrophobic, and the like). “Function-conservative”variants also include any polypeptides that have the ability to elicitantibodies specific to a designated polypeptide.

In general, nucleic acid manipulations used in practicing the presentinvention employ methods that are well known in the art, as disclosedin, e.g., Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook,Fritsch and Maniatis, Cold Spring Harbor) and Current Protocols inMolecular Biology (Eds. Ausubel, Brent, Kingston, More, Feidman, Smithand Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY, N.Y., 1997).

The present invention is directed to methods and compositions forkilling Gram-positive bacteria that take advantage of the bactericidalaction of Group IIA phospholipase A2 (PLA2). In practicing theinvention, bacteria are exposed to or contacted with, a Gram-positivebactericidal-effective amount of mutant human Group IIA PLA2, resultingin the rapid inactivation and death of Gram-positive bacteria. Accordingto the invention, bacterial infections in humans can be treated byadministering mutant human Group IIA PLA2. The invention alsoencompasses pharmaceutical formulations suitable for therapeuticadministration.

The mutant human Group II A PLA2 of the present invention has 10×greater antibacterial activity and a net charge +2 greater than thehuman WT homologue (+17 vs. +15). Based on the comparison of thestructural and functional properties of rabbit and human wild-type andmutant Group II A PLA2 (see FIG. 1), discrete alterations of human GroupII A PLA2 by site-specific mutagenesis yielded novel human Group II APLA2 proteins (herein referred to as “mutant”) with markedly enhancedantibacterial activity which may be measured using any procedurewell-known in the art, including that described in Example 3 below.

Previous studies with site-specific mutants of human Group IIA PLA2indicated that the high net (+) charge of Group IIA PLA2 in addition toits catalytic properties were essential for the potent antibacterialproperties of Group IIA PLA2. The high net (+) charge and antibacterialproperties are unique attributes of this subset of PLA2. In addition,comparison of the structural and functional properties of native andrecombinant rabbit and human Group IIA PLA2 demonstrated higherantibacterial activity in the rabbit PLA2 along with a higher net (+)charge (+17 for the rabbit enzyme, +15 for the human PLA2). Followingcloning of the cDNA encoding rabbit Group IIA PLA2 and sequenceanalysis, comparison of the primary structures of rabbit and human GroupIIA PLA2 revealed 32 sites that were different in these two enzymes (26%of 124 residues; no gaps). Of these, at least 22 represented relativelyconservative substitutions. Two differences stood out; arginine (rabbit)vs. glycine (human) at residue 72 and lysine (rabbit) vs. threonine(human) at residue 103. These differences account for the higher netcharge of the rabbit PLA2 and were located along a highly cationic ridgeon the enzyme surface (see FIG. 1). Mutagenesis studies indicated thatthe charge properties of this region are essential for potentantibacterial activity and also suggested that the charge properties ofother regions may not be equally important for antibacterial activity.Consequently, residues 72 and 103 were chosen for mutagenesis. It ispossible that introduction of basic residues either at other surfacesites within this highly cationic ridge or outside this region, even atsites that do not normally contain basic amino acids in the native GroupIIA PLA2, could also confer increased antibacterial activity.

It should be noted that among all the “low Mr” (i.e. 13-18 kDa) PLA2,including the native Group IIA PLA2, the density and distribution ofcharged residues along the enzyme surface varies widely withoutaffecting overall protein conformation and catalytic activity towardartificial substrates. This is also true in surface charge changesintroduced genetically in PLA2 variants. Thus, great variation in theenzyme surface charge can be well tolerated, affording ample opportunityto create many permutations of enzyme structure that may enhanceantibacterial potency.

In a preferred embodiment, E. coli is stabily transformed with a cDNAencoding mutant human Group IIA PLA2, as set forth in FIG. 3A and isused as a recombinant source of Group IIA PLA2 (see Example 1). Forrecombinant expression, Group IIA PLA2-encoding DNA, contained within aDNA vector, must be operably linked to a transcriptional promoter sothat functional Group IIA PLA2 mRNA is transcribed and Group IIA PLA2protein is synthesized within the transformed host cell. Preferably, therecombinant protein is recovered in E. coli inclusion bodies and thenthe expressed protein is subjected to in vitro reversibledenaturation/reduction followed by renaturation/oxidation to promoteproper disulfide bond formation.

The invention also encompasses vectors comprising mutant humanPLA2-encoding sequences, cells comprising the vectors, and methods forproducing mutant human PLA2 that involve culturing the cells.

A large number of vectors, including plasmid and fungal vectors, havebeen described for expression in a variety of eukaryotic and prokaryotichosts. Advantageously, vectors may also include a promotor operablylinked to the mutant human PLA2 encoding portion. The encoded mutanthuman PLA2 may be expressed by using any suitable vectors and hostcells, using methods disclosed or cited herein or otherwise known tothose skilled in the relevant art. The particular choice of vector/hostis not critical to the invention.

Vectors will often include one or more replication systems for cloningor expression, one or more markers for selection in the host, e.g.antibiotic resistance, and one or more expression cassettes. Ligation ofthe coding sequences to the transcriptional regulatory sequences may beachieved by known methods. Suitable host cells may betransformed/transfected/infected by any suitable method includingelectroporation, CaCl₂ mediated DNA uptake, fungal infection,microinjection, microprojectile, or other established methods.

Appropriate host cells include bacteria, archebacteria, fungi,especially yeast, and plant and animal cells, especially mammaliancells. Of particular interest are E. coli, B. subtilis, S. cerevisiae,SF9 cells, C129 cells, 293 cells, Neurospora, CHO cells, COS cells, HeLacells, and immortalized mammalian myeloid and lymphoid cell lines.Preferred replication systems include M13, ColE1, SV40, baculovirus,lambda; adenovirus, and the like. A large number of transcriptioninitiation and termination regulatory regions have been isolated andshown to be effective in the transcription and translation ofheterologous proteins in the various hosts. Examples of these regions,methods of isolation, manner of manipulation, etc. are known in the art.Under the appropriate expression conditions, host cells can be used as asource of recombinantly produced mutant human PLA2.

Nucleic acids encoding mutant human PLA2 polypeptides may also beintroduced into cells by recombination events. For example, such asequence can be introduced into a cell, and thereby effect homologousrecombination at the site of an endogenous gene or a sequence withsubstantial identity to the gene. Other recombination-based methods,such as non-homologous recombinations or deletion of endogenous genes byhomologous recombination, may also be used.

The invention also encompasses isolated and purified mutant human PLA2polypeptides, including, e.g., a polypeptide having the amino acidsequence depicted in FIG. 3B, as well as function-conservative variantsof this polypeptide, including fragments that retain antibacterialactivity as described above.

Purification of mutant human Group IIA PLA2 from recombinant sources maybe achieved by methods well-known in the art, including withoutlimitation ion-exchange chromatography, reversed-phase high performanceliquid chromatography (HPLC) on C4 columns, gel filtration, isoelectricfocusing, affinity chromatography, immunoaffinity chromatography, andthe like. For some purposes, it is preferable to produce the polypeptidein a recombinant system in which the protein contains an additionalsequence tag that facilitates purification, such as, but not limited to,a polyhistidine sequence. The polypeptide can then be purified from acrude lysate of the host cell by chromatography on an appropriatesolid-phase matrix.

In a preferred embodiment, a cell-free fluid containing mutant humanGroup IIA PLA2 is subjected to ion-exchange chromatography onSP-Sepharose, followed by reversed-phase high performance liquidchromatography (HPLC) on a C4 column. Purity of the final Group IIA PLA2preparations is confirmed by SDS-PAGE and by OD at 280 m, using theknown extinction coefficient for this protein (OD of 1.0=0.9 mg/ml).

The isolated polypeptide may be modified by, for example,phosphorylation, sulfation, acylation, or other protein modifications.It may also be modified with a label capable of providing a detectablesignal, either directly or indirectly, including, but not limited to,radioisotopes and fluorescent compounds.

According to the present invention, the mutant human Group IIA PLA2 ofthe present invention is characterized by bactericidal activity againstgram positive bacteria, such as S. aureus (see Example 3). Antibacterialactivity of mutant human Group IIA PLA2 may be quantified by measuringthe colony-forming ability of susceptible bacteria that have beenincubated with or without increasing amounts of mutant human Group IIAPLA2. Typically, a suspension of 10⁶ bacteria/ml (e.g., S. aureus ) isexposed to 1-500 ng/ml of mutant human Group IIA PLA2 for 60-90 minutesat 37° C., after which the cells are mixed with molten agar and plated.After overnight growth, bacterial colonies are compared between mutantGroup IIA PLA2-treated and untreated cultures.

The present invention encompasses, in addition to the mutant human GroupIIA PLA2 disclosed herein, other recombinant forms of Group IIA PLA2that are formed by site-specific genetic manipulations and havedetectable Gram-positive bactericidal activity. The methods andcompositions of the present invention encompass any deletion, addition,or substitution mutant of Group IIA PLA2 produced by the methodsdescribed herein that increase the wild-type enzymatic and antibacterialactivity.

It will be understood that the methods for expression, purification, andactivity measurements described above for the mutant human Group IIAPLA2 can also be applied to variant Group IIA PLA2 species. Thus also,only routine experimentation is required to identify additional, newuseful Group IIA PLA2 variants.

Therapeutic Applications

The enhanced antibacterial potency of the mutant human Group II A PLA2protein described herein provides new therapeutic approaches to thetreatment of potentially life-threatening infections caused bymulti-drug resistant Gram-positive bacteria. The applications includewound and bloodstream infections with methicillin-resistant S. aureus(MRSA) and nosocomial infections with vancomycin-resistant Enterococcusfaecium. These infections are much more common and potentially lifethreatening in immunocompromised or hospitalized patients. The abilityof the Group II A PLA2 to act in synergy with various β-lactamantibiotics and, most importantly, with otherwise β-lactam-resistantbacteria (e.g. MRSA), also allows for the use of the PLA2 of the presentinvention in conjunction with antibiotics otherwise rendered ineffectiveby the growing prevalence of antibiotic resistance.

According to the present invention, recombinant mutant human Group IIAPLA2 may be formulated with a physiologically acceptable carrier, suchas, for example, phosphate buffered saline or deionized water. Thepharmaceutical formulation may also contain excipients, includingpreservatives and stabilizers, that are well-known in the art. Thecompounds can be formed into dosage units such as, for example, liquids,tablets, capsules, powders, suppositories, and may additionally includeexcipients that act as lubricant(s), plasticizer(s), colorant(s),absorption enhancer(s), bactericide(s), and the like. The dosage formsmay contain mutant human Group IIA PLA2 at concentrations rangingbetween about 100 ng/ml and about 100 μg/ml. Solid dosage forms such astablets and powders may contain the mutant human Group IIA PLA2 of thepresent invention at appropriate concentrations so that bactericidaleffective amounts of mutant human Group IIA PLA2 (see below) can bedelivered using conventional administration regimens. It will beunderstood that the pharmaceutical formulations of the present inventionneed not in themselves contain the entire amount of the agent that iseffective in treating the disorder, as such effective amounts can bereached by administration of a plurality of doses of such pharmaceuticalformulations.

Modes of administration of the mutant human Group IIA PLA2 of thepresent invention to achieve a therapeutic benefit include topical, oraland enteral, intravenous, intramuscular, subcutaneous, transdermal,transmucosal (including rectal and buccal), and by-inhalation routes.Generally, Group IIA PLA2 and specifically the mutant human Group IIAPLA2 of the present invention are extremely stable proteins and toleratea wide variety of environmental conditions. It will be understood thatthe mode of administration will depend on the nature of the syndrome,including the location. and severity of the Gram-positive bacterialinfection. For example, skin lesions may be treated using a topicalointment, whereas a bacteremia may require intravenous administration.An internal but localized infection may be treated by injecting theformulation directly into the site of the infection.

An “effective amount” of the mutant human Group IIA PLA2 of the presentinvention for treating a particular bacterial infection is an amountthat results in a detectable reduction in the severity of the infection.This may be measured directly, i.e., by counting or culturing thepathogenic microorganisms, or indirectly, by monitoring clinical signsof infection, such as fever or purulent discharge. Typically,administration of the mutant human Group IIA PLA2 will result in thelessening or amelioration of at least one symptom of the infection. Anyamelioration resulting from administration of the mutant human Group IIAPLA2 of the present invention of any symptom of infection is within thescope of the invention. The effective amount for treating a givensyndrome in a human can be determined by routine experimentationwell-known in the art, such as by establishing a matrix of dosages andfrequencies and comparing a group of experimental units or subjects toeach point in the matrix.

An additional consideration in establishing the optimum dosage of themutant human Group IIA PLA2 for treating bacterial infections ispotential toxicity. Though there have been reports that Group IIA PLA2possesses inflammatory activity (see, for example, Bomalaski et al., JImmunol. 146:3904, 1991; and Cirino et al., J Rheumatol. 21:824, 1994),this phenomenon was only observed at Group IIA PLA2 concentrationsseveral orders of magnitude higher than those at which bactericidaleffects are observed. Furthermore, the preparations used in the studiescited above were contaminated with endotoxin, which itself has a potentinflammatory activity. Thus, without wishing to be bound by theory, itis believed that bactericidal effective amounts of Group IIA PLA2 can beadministered to humans without causing inflammatory or other detrimentalside effects. In addition, it is preferable to treat humans with GroupIIA PLA2 derived from the same species.

The effective amount of the mutant human Group IIA PLA2 of the presentinvention for treating infections caused by gram-positive bacteria to beadministered may range between about 1 and about 100 μg/kg/body weight,preferably from about 1 to about 10 μg/kg/body weight. In a preferredembodiment, mutant human Group IIA PLA2 of the present invention isformulated in a sterile saline solution, which is administeredintravenously to a patient suffering from an antibiotic-resistant S.aureus bacteremia.

In another embodiment, an antibacterial formulation is preparedcontaining, in addition to the mutant human Group IIA PLA2, otherconventional antibiotics (such as, for example, β-lactam antibiotics),or other bioactive substances, that may act additively orsynergistically with the mutant human Group IIA PLA2 of the presentinvention to kill Gram-positive bacteria. It is believed that the use offormulations containing the mutant human Group IIA PLA2 of the presentinvention in conjunction with, for example, sub-lethal doses of otherantibiotics would provide a clinical advantage in reducing the overalladministration of antibiotics (thus lessening the development ofantibiotic-resistant strains).

Antibiotics that can be used in conjunction with mutant human Group IIAPLA2 of the present invention in the methods and compositions of thepresent invention include without limitation penicillins (such asampicillin, amoxicillin, oxacillin, and the like), cephalosporins,aminoglycosides (such as streptomycin, neomycin, kancmycin, gentamicin,and the like), tetracyclines, chloramphenical, and vancomycin.Commercial sources for each of these are presented in Table I below.

TABLE 1 Drug Source City, State Ampicillin Warner Chilcott LaboratoriesRockaway, NJ Amoxicillin Warner Chilcott Laboratories Rockaway, NJOxacillin Teva Pharmaceuticals Sellersville, PA Cefotaxime HoechstMarion Roussel Kansas City, MO Streptomycin Pfizer New York, NY NeomycinMerck West Point, PA Kanamycin SolePak Pharmaceuticals Boca Raton, FLGentamicin SoloPak Pharmaceuticals Boca Raton, FL Tetracycline LederleStandard Products Philadelphia, PA Chloramphenicol Fujisawa Deerfield,IL Vancomycin Eli Lilly and Co. Indianapolis, IN

As shown below in Example 7, the human mutant Group IIA PLA2 of thepresent invention was effective in treating S. aureus infection in vivo.Given the rapidly spreading occurrence of drug resistant bacteria, themutant enzyme of the present invention will provide a useful addition tothe arsenal of presently used anti-Gram positive antibiotics.

The following examples are intended to further illustrate the inventionwithout limiting the scope thereof.

EXAMPLE 1

Preparation of Mutant (G72K.T103K) Human Group II A PLA2 cDNA

cDNA encoding the mutant human Group II A phospholipase A2 was producedby mismatched primer PCR using oligonucleotide primers directed againstwild-type human Group II PLA 2 cDNA (clone hp PLA2 9-1) subcloned withinpEE14 (CellTech) as described in Weiss, et al., 1994, J. Biol. Chem.269: 26331-26337. The oligonucleotide primer 5′TTTGCTAGAAACAAGAAGACCTACAAT 3′ SEQ. ID NO:3 has a single base pairchange (underlined and in bold) conferring the threonine to lysinesubstitution at residue 103 and is complementary to codons 98-106 of thenon-coding strand of the human Group II A phospholipase A2. The singlemutant T103K was created by substitution of lysine for threonine atresidue 103. The double mutant G72K.T103K was constructed on the DNAtemplate of the T103K mutant using the primer 5′TTTAGCAACTCGAAGAGCAGAATCACC 3′ SEQ. ID NO:4 which is complementary tothe non-coding strand from residues 68-76 with the indicated two basechange to produce the glycine to lysine substitution at residue 72.Furthermore, to facilitate purification of recombinant Group II A PLA2,expressed as part of a fusion protein, one additional substitution wasintroduced (L8M) using the oligonucleotide primer 5′GCTCGAGATGAATTTGGTGAATTTCCACAGACTGATC 3′ SEQ. ID NO:5 which iscomplementary to the non-coding strand of the human Group II A PLA2 fromcodons 1-9. This alteration eliminates the single methionine amino acidresidue within the PLA2 coding region permitting excision of intact PLA2from the fusion partner by CNBr treatment.

EXAMPLE 2

Expression and Purification of Mutant (G72K.T103K) Human Group IIA PLA2

The mutated Group II A PLA2 cDNA was subcloned into Xho I and EcoRIrestriction sites of E. coli expression vector pRSETA (Invitrogen) andexpressed as a fusion protein under the control of the bacteriophage T7promoter. Expression of the recombinant protein was induced by thetreatment of transformed E. coli BL21 (DE3) with IPTG. The recombinantprotein was recovered in inclusion bodies, extracted and modified byS-sulfonation as previously described. (Thannhauser et al., Biochemistry24 7681-7688., 1985; Liang, N. S. et al. FEBS Letters 334:55-59. 1993;Fourcade et al., Cell 80: 919-923, 1995). The S-sulphonated protein wasprecipitated by dialysis against 1% acetic acid and cleaved by cyanogenbromide to release mature Group IIA PLA2 from the fusion protein.Refolding and disulfide bond formation of recombinant Group IIA PLA2 wascarried out in the cold for 72 hours in 10 mM sodium borate buffer (pH8.5) containing 2 mM cystine, 10 mM cysteine, 10 mM CaCl₂ and 0.85 Mguanidinium hydrochloride followed by dialysis against 50 mM sodiumacetate/acetic acid buffer, pH 5.0. Refolded, active Group IIA PLA2 waspurified from improperly folded, inactive protein by chromatography onSP-Sepharose and reversed-phase high performance liquid chromatography(HPLC) on a C4 column. The purity of the recovered Group IIA PLA2 wasconfirmed by analytical reversed-phase HPLC and by absorbence at 280 nm,using the known extinction coefficient for this protein (OD of 1.0=0.9mg/ml).

EXAMPLE 3

Comparison of the Bactericidal Activity of Wild-type and RecombinantHuman, and Rabbit Group IIA PLA2s Against Staphylococcus aureus 52A

The bactericidal activity of wild-type human and recombinant mutanthuman and rabbit Group IIA PLA2s against Staphylococcus aureus 52A (10⁶per ml)was determined. S. aureus was incubated with 0.01-100 nM of eachGroup IIA PLA2 and incubated at 37° C. for 2 hours in RPMI-1640 mediumcontaining 10 mM HEPES, pH 7.4 and 1% (w/v) albumin. After incubation,bacterial viability was determined by measuring the colony formingability of the bacteria in trypticase soy agar. Bacterial colonies wereenumerated after 18-24 hours at 37° C. The results are expressed as thepercentage of the colony forming units (CFU) of the original bacterialinoculum (i.e. at T=0).

The graphical results depicted in FIG. 2 show that about 10-fold lowerconcentrations of rabbit PLA2 than of human PLA2 suffice to produce thesame reduction of CFU. Therefore, rabbit Group IIA PLA2 had a greaterbactericidal effect on S. aureus cells than the human enzyme.

EXAMPLE 4

Comparison of the Bactericidal Activity of Wild-type (WT) andRecombinant Human, and Rabbit Group II A PLA2 Toward S. aureus RN450

The bactericidal activity of wild-type and recombinant mutant human andrabbit Group IIA PLA2s against Staphylococcus aureus RN450 wasdetermined.

S. aureus RN450 (10⁶/ml) was incubated with 1-500 ng/ml of each GroupIIA PLA2 at 37° C. for 60 minutes in RPMI-1640 medium containing 10 mMHEPES, pH 7.4 and 1% (w/v) albumin. Bacterial viability was determinedas in Example 3.

The graphical results depicted in FIG. 4 show that the bactericidalactivities of recombinant mutant human Group IIA PLA2 and WT rabbitGroup IIA PLA2 are nearly equivalent. Furthermore, the geneticallymodified mutant human Group IIA PLA2 had increased antibacterialactivity against S. aureus compared to wild-type human Group IIA PLA2.

EXAMPLE 5

Comparison of the Bactericidal Activity of Recombinant Human and RabbitGroup II A PLA2 Toward S. aureus RN450 and Two Clinical Isolates,Strains 5A and 18

S. aureus RN450 and two clinical isolates, strains 5A (MRSA) and 18(10⁷/ml were incubated with 1-500 ng/ml of each Group IIA PLA2 at 37° C.for 90 minutes in 70% (v/v) pooled human sera diluted in Hanks' balancedsalts solution and buffered with 10 mM HEPES, pH 7.4.

As shown in FIGS. 4 and 5, the mutant human Group IIA PLA2 wasessentially as active as the wild-type rabbit enzyme toward each of theseveral strains of S. aureus, and, hence, significantly more active thanthe wild-type human Group IIA PLA2. This is manifested in both anartificial laboratory medium (FIG. 4) and in an environment that moreclosely simulates that of circulating body fluids (FIG. 5).

These findings further demonstrate that the novel mutant Group IIAphospolipase A2 described herein represents a more potent antibacterialproduct than the wild-type human enzyme.

EXAMPLE 6

Encapsulated and Non-encapsulated S. aureus are Killed by Group IIA PLA2

One setting in which mobilization of extracellular Group IIA PLA2 may beparticularly important is against encapsulated strains of S. aureus. Themajority of bacteremic isolates of S. aureus are encapsulated(Hochkeppel et al., J. Clin. Microbiol. 25:526-530) and bacteriarecovered from more chronically infected sites as in cystic fibrosis arealso covered with an extracellular carbohydrate polymer (McKenney etal., Science 284:1523-1527). In the absence of type-specificanti-capsular antibodies, encapsulated strains are relatively resistantto opsonophagocytic killing by PMN (Xu et al., Infect. Immun.60:1358-1362; Table I). In contrast, purified Group IIA PLA2 and PLA2present in elicited inflammatory fluids exhibit equipotent bactericidalactivity against encapsulated S. aureus and isogenic non-encapsulatedderivates (FIGS. 6, 7). Moreover, despite inability of PMN withinexperimentally elicited acute inflammatory exudates to efficientlyingest encapsulated S. aureus (Table 2), the bacteria are stillefficiently killed by the inflammatory exudates in an extracellular andPLA2-dependent fashion (FIG. 8). Thus, Group IIA PLA2 can provide apotent extracellular weapon against phagocytosis-resistant encapsulatedbacteria that is fully active in inflammatory exudates.

TABLE 2 EFFECT OF CAPSULE ON SUSCEPTIBILITY OF S. AUREUS TO PHAGOCYTOSISBY RABBIT PMN Strain Bacteria/100 PMN 1B (non-encapsulated) 43.5 1C(encapsulated) 2.3

S. aureus type 1C (encapsulated) and an isogenic non-encapsulatedderivative (type 1B) were incubated for 30 min. with rabbit pentanealexudate PMN at bacteria/PMN ratio of 2:5 (10⁶ bacteria and 2.5×10⁶PMN/ml). At end of incubation, suspensions were diluted, smears preparedby cytospin and stained. PMN-associated bacteria were visualized bylight microscopy and counted. Results are expressed as number ofbacteria associated with PMN/100 PMN counted. Results indicate a 100%uptake of non-encapsulated bacteria and <10% uptake of encapsulatedbacteria. Similar results were obtained when incubations were carriedout in HEPES-bufferred Hanks' balanced salt solution or in PLA₂-depletedascitic fluid.

EXAMPLE 7

Animal Experiment to Test Efficacy of Administered Human Mutant GroupIIA PLA2 Against S. aureus Infection in vivo

Materials and Methods

In the example presented below, the following materials and methods wereused.

Animals: CD/1 mice

Bacteria: Reynolds strain of Staphylococcus aureus (encapsulated; grownovernight on Columbia agar to maximize encapsulation as in experimentsshown in Example 6).

Administration of bacteria: 2×10⁷/ml in 0.2 ml of sterile RPMIsupplemented with 10 mM HEPES (pH 7.4) and 1% bovine serum albumin.Intraperitoneal (i.p.) inoculation.

Adminstration of PLA2: 15 μg/0.2 ml of above medium i.p. approx. 10-15min after inoculation of bacteria. Control animals received mediumalone. Enzyme administered was Mutant [G72K.T103K] Group IIA PLA 2.

Assays of course of infection:

a) @ 30, 60, 120, and 240 min after infection, blood samples taken fromtail vein from each of 4 animals in control and PLA2-treated groups.There were a total of 16 animals in each group; each animal was bledonly once in this time period. Levels of bacteremia were assessed bymeasurement of bacterial CFU in blood.

b) @ 1 day after infection, 8 animals from each group were sacrificed.Blood was collected again to measure bacteremia and the peritonealcavity was washed and inspected to look macroscopically for abscesses(none seen) and measure intraperitoneal bacteria by assay of CFU.

c) @ 6 days after infection, the remaining 8 animals from each groupwere sacrificed and infection in blood and peritoneal cavity wasmeasured as above (still no abscesses seen). This animal model produceslocal and disseminated infection in control animals that is eventuallyself-limiting. The level of metastatic infection is greatest at approx.6 days. In addition, kidneys were excised, weighed (no significantdifference between animals within and between treatment groups) andhomogenized to facilitate assay of bacterial CFU within infected kidney(i.e. representing metastatic infection). Abscesses were seen in two of16 control animals; none were seen in PLA2-treated animals.

The results are shown in FIGS. 9, 10 and 11 for b, a and c,respectively, above.

5 1 375 DNA Homo sapiens 1 aatttggtga atttccacag actgatcaag ttgacgacaggaaaggaagc cgcactcagt 60 tatggcttct acggctgcca ctgtggcgtg ggtggcagaggatcccccaa ggatgcaacg 120 gatcgctgct gtgtcactca tgactgttgc tacaaacgtctggagaaacg tggatgtggc 180 accaaatttc tgagctacaa gtttagcaac tcgaagagcagaatcacctg tgcaaaacag 240 gactcctgca gaagtcaact gtgtgagtgt gataaggctgctgccacctg ttttgctaga 300 aacaagaaga cctacaataa aaagtaccag tactattccaataaacactg cagagggagc 360 acccctcgtt gctga 375 2 124 PRT Homo sapiens 2Asn Leu Val Asn Phe His Arg Leu Ile Lys Leu Thr Thr Gly Lys Glu 1 5 1015 Ala Ala Leu Ser Tyr Gly Phe Tyr Gly Cys His Cys Gly Val Gly Gly 20 2530 Arg Gly Ser Pro Lys Asp Ala Thr Asp Arg Cys Cys Val Thr His Asp 35 4045 Cys Cys Tyr Lys Arg Leu Glu Lys Arg Gly Cys Gly Thr Lys Phe Leu 50 5560 Ser Tyr Lys Phe Ser Asn Ser Lys Ser Arg Ile Thr Cys Ala Lys Gln 65 7075 80 Asp Ser Cys Arg Ser Gln Leu Cys Glu Cys Asp Lys Ala Ala Ala Thr 8590 95 Cys Phe Ala Arg Asn Lys Lys Thr Tyr Asn Lys Lys Tyr Gln Tyr Tyr100 105 110 Ser Asn Lys His Cys Arg Gly Ser Thr Pro Arg Cys 115 120 3 27DNA Artificial Sequence oligonucleotide primer 3 tttgctagaa acaagaagacctacaat 27 4 27 DNA Artificial Sequence oligonucleotide primer 4tttagcaact cgaagagcag aatcacc 27 5 37 DNA Artificial Sequenceoligonucleotide primer 5 gctcgagatg aatttggtga atttccacag actgatc 37

What is claimed is:
 1. A method for killing Gram-positive bacteria in a human patient which comprises contacting said bacteria with a bactericidal-effective amount of mutant human Group IIA phospholipase A2 (Group IIA PLA2) having SEQ ID NO:2.
 2. The method of claim 1, wherein said bacteria are selected from the group consisting of Micrococcus, Staphylococcus, Streptococcus, Peptococcus, Peptostreptococcus, Enterococcus, Methanobacterium, Bacillus, Clostridium, Lactobacillus, Listeria, Erysipelothrix, Corynebacterium, Propionibacterium, Eubacterium, Actinomyces, Arachnia, Bifidobacterium, Bacterionema, Rothia, Mycobacterium, Nocardia, Streptomyces, and Micropolyspora.
 3. The method of claim 1, wherein said bacteria are contacted with between about 1 and about 100 μg per kg body weight of said human patient of mutant human Group IIA PLA2.
 4. The method for killing Staphylococcus aureus bacteria which comprises contacting said bacteria with a bactericidal-effective amount of mutant Group IIA PLA2 having SEQ ID NO:2.
 5. A method for treating a Staphylococcus aureus infections in a human which comprises administering to said human an amount effective for treating said infection with mutant human Group IIA PLA2 having SEQ ID NO:2.
 6. The method of claim 5 wherein said amount effective for treating said infection ranges between about 1 and about 100 μg/kg body weight of said human.
 7. A method for treating a human patient suffering from an infection caused by a Gram-positive bacteria comprising administering to a human in need of such treatment: (a) mutant human Group IIA PLA2 having SEQ ID NO:2, and (b) an antibiotic, the amounts of (a) and (b) together being effective to treat said infection.
 8. The method of claim 7, wherein said antibiotic is selected from the group consisting of ampicillin, amoxicillin, oxacillin, cephalosporins, streptomycin, neomycin, kancmycin, gentamicin, tetracyclines, chloramphenical, and vancomycin. 